Optical fibre confocal imager with variable near-confocal control

ABSTRACT

A confocal imaging system using optical fibers is provided which has a flexible near confocal optical transmission means having a light collection end adjacent to a light collection of the confocal optical transmission means and adapted to transmit only near confocal light emerging from points in the object located within a range of distances above and below the focal plane, in such a manner that a selected portion of the near confocal light emerging from greater than any selected distance within the range is substantially separable from the remainder. The system also has variable selection means to exclude from detection the selected portion.

FIELD OF THE INVENTION

This invention relates to confocal imaging systems which use a flexibleoptical transmission means such as optical fibres as a substitute forthe return pinhole, and more particularly but not limited to confocalmicroscopes constructed using optical fibres.

DESCRIPTION OF THE PRIOR ART

Confocal microscopy can be considered to have originated with the workof Marvin Minsky. His U.S. Pat. No. 3,013,467 describes a system inwhich light passes through a pinhole, traverses a beamsplitter and isfocused by an objective to a spot on or within a specimen. In anepi-illumination embodiment, light returning from the spot region isconverged by the same objective lens, reflected by its second encounterwith the beamsplitter and passes through a second pinhole to a photodetector. The geometry of the arrangement is such that the focused spot(Gaussian waist volume) is the only volume within the specimen fromwhich the general set of ray paths returning through the lens willretrace their outgoing paths to pass through the second pinhole to thephoto detector. Light reflected from above or below this focus whichpasses through the objective lens will be largely blocked off by theopaque sheet material forming the pinhole surrounding area.

The electrical signal from the photo detector will give a value for thelight reflected from the spot. If the specimen is moved the changes inelectrical output from the photo detector indicate changes in the levelof light return from the material of the specimen along the path of thespot. If the specimen is moved in a two dimensional raster then a twodimensional rastered map of the return light intensity can be build upbased on the raster synchronous modulation of the electrical output fromthe photo detector. This can be displayed on a cathode ray screen or byother means giving an image which is a sharp optical slice,substantially eliminating the contribution of light from above or belowthe focal plane. Such light normally reduces the contrast and blurs theimage in conventional microscopy, particularly from translucentbiological specimens, and renders high power microscopic observation ofthick tissue sections impossible. The use of an optical fibre in theplace of one or more of the pinholes is disclosed in U.S. Pat. No.5,120,953. In such a fibre confocal microscope, the core of the opticfibre effectively acts as the pinhole, and, when the fibre is singlemoded, the light leaves the fibre as a single set of concentricexpanding wavefronts and the system becomes diffraction limited andmaximum resolution is obtained.

The chief advantage of using a fibre to replace the pinhole is that thetwo sides of the pinhole are effective optically connected by the coreof the fibre, but are physically independent and can be independentlyand separately positioned. The major advantages conferred by this are

(a) the several large and heavy components of the microscope can belocated in any convenient position and do not need to be rigidly locatedwith respect to the specimen;

(b) the fibre tip itself can be mechanically scanned to give the rasterrequired to build-up the image data set;

(c) an "in fibre" evanescent wave beamsplitter can be employed.

A disadvantage of existing fibre confocal microscopes is that for thesesystems there is no direct functional equivalent of opening up thepinhole. Most bulk optic laser scanning confocal microscopes include afunction in which the second pinhole can be progressively enlarged.While for the purposes of the highest resolution image, the smallestsized second pinhole is desirable consistent with a reasonable opticalsignal strength, in practice it is desirable to enlarge and contract thesecond pinhole as the microscopist examines the object, by the means ofoperating a continuously variable diaphragm. The diaphragm opens theaperture and collects an increasing fraction of light from the doublecone volume on either side of the Gaussian waist region. This increasesthe strength of the electrical signal but at the expense of opticalresolution. This procedure is used

(a) during a "search mode" in the early stages of observation wherequick single scan images are being used to locate the desired structure;

(b) where a rapidly moving structure is to observed which is notrepetitive and thus cannot be scan synchronised;

(c) where an increased depth of field is desired for large depth threedimensional reconstructions;

(d) where the fluorescence or the reflection signal is very weak;

(e) where the fluorophore is fugitive (ie. easily photobleached orspontaneously decomposing).

SUMMARY OF THE INVENTION

It is an object of the current invention to be able to construct a fibreoptic confocal imaging system which retains the advantages of the use ofthe optical fibre but in addition has an equivalent function of openingup and closing down a pinhole without the necessity of providing aphysical pinhole or diaphragm adjacent to the specimen optics.

Therefore in accordance with a broad aspect of the invention there isprovided a confocal imaging system comprising:

a light source for supply of a light beam;

light focusing means for focusing light from the beam onto a pointobservational field on or within an object and for receiving objectemanated light emanating from the vicinity of the point observationalfield;

a detector for detecting the object emanated light;

scanning means operable to cause relative movement between the objectand the point observational field such that the point observationalfield scans over a focal plane transverse to an optical axis of theimaging system; and

flexible optical transmission means for transmitting the source lightbeam from the light source to the light focusing means and fortransmitting the object emanated light to the detector, and having lightseparator means to separate the object emanated light from the lightbeam for passing to the detector and confocal optical transmission meansto transmit the object emanated light emerging only from the pointobservational field;

wherein the optical transmission means further comprises

(i) flexible near confocal optical transmission means having a lightcollection end adjacent to a light collection end of the confocaloptical transmission means and adapted to transmit only near confocallight emerging from points in the object located within a range ofdistances above and below the focal plane in such a manner that aselected portion of the near confocal light emerging from greater than acorresponding selected distance within said range is substantiallyseparable from the remainder; and

(ii) an exit region for exit of at least a portion of said near confocallight from the flexible near confocal optical transmission means;

and wherein there is further provided variable selection means to definesaid selected portion and exclude it from the detector.

By providing separable transmission through flexible and selectablemeans, a variable pinhole effect can be provided which may be locatedremotely of the specimen.

In one class of embodiment, the near confocal optical transmission meanscomprises a plurality of optically isolated channels having adjacentends at said light collection end to provide said substantiallyseparable transmission. The plurality of channels may be provided by abundle of optical fibres, or a large diameter optical fibre withmultiple cores. Alternatively, the plurality of channels may be aplurality of coaxial concentric waveguides, mutually separated byoptically insulating material.

In this first class of embodiments, an exit region of the near confocaloptical transmission means may be provided by a plurality of etchedsections of fibre exposing different ones or subsets of said pluralityof channels and containing optical cement to divert light travelling inthe corresponding one or subset of channels to corresponding photodetectors. In this case, the variable selection means comprisesswitching circuitry or the like to select output from different photodetectors. Alternatively, the exit region may be provided by oppositeends of the plurality of isolated channels forming an emission end ofthe fibre or fibre bundle, and the variable selection means may comprisefocusing means to project an image of the emission end onto a regioncontaining a variable diaphragm to progressively exclude from detectionsaid selected portion, the detector being disposed behind the diaphragm.

In a second class of embodiments, the near confocal optical transmissionmeans comprises a wide diameter fibre or the cladding of a single modeoptical fibre. In this class of embodiments, the substantialseparability of said selected portions may be attained if the focussingmeans causes rays entering the light collection end of optical fibre tobe transmitted through the fibre at an angle which increases with thedistance of a point of entry of the ray into the collection end from theoptical axis of the fibre. The variable selection means may include avariable diaphragm disposed adjacent the exit region of the opticalfibre to exclude light emerging at greater than a selected angle.

In embodiments where the exit region is provided by an emission end ofthe fibre or fibre bundle, the variable selection means may also includenear confocal focussing means to focus an image of the emission end ofthe fibre onto a second variable diaphragm.

In other embodiments of the second class the exit region is provided byan exposed side of the fibre such as an area of the fibre stripped ofits outer jacket and contacting a material with refractive indexsuitably matched to the fibre so as to extract the near confocal light.The near confocal light may be extracted from a single such region andthe variable selection performed by variable diaphragm means.Alternatively, the near confocal light may be extracted from a pluralityof regions along the length of the fibre contacting materials havingprogressively greater refractive index to progressively extract rays oflower angle, the variable selection means comprising optical orelectronic switching means.

In a third class of embodiments, the near confocal optical transmissionmeans comprises a gradient index fibre. In this class, the exit regionmay be provided by successively deepening etched areas in the fibre sidewith corresponding photo detectors. Alternatively, the exit region maybe provided by an emission end of the fibre. In such cases, a firstvariable diaphragm may be provided to admit only low angle light throughnear confocal focussing means to project an image of the fibre tip ontoa second variable diaphragm in front of the detector.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order that the invention may be more clearly ascertained, preferredembodiments will now be described with reference to the accompanyingdrawings, in which

FIG. 1 is a diagram of ray paths emerging from the vicinity of the pointobservational field of a confocal microscope and being focused onto thecollection end of an optical transmission means;

FIGS. 2a, 2b, 2c and 2d are examples of the plurality of isolatedchannels of the first embodiment of the invention;

FIG. 3 is a diagram showing a schematic optical arrangement of avariation of the first embodiment using "four leaved clover" fibres (see26) as the near confocal optical transmission means, and etched regionsof fibre to provide the progressive selection;

FIG. 4 is another variation of the first embodiment showing the use of aconcentric wave guide structure;

FIG. 5 is another variation of the first embodiment showing theprogressive selection means provided by the projection of an image of anemission end of the fibre;

FIG. 6 is diagram showing the principle of transmission of light raysthrough the cladding of a single mode optical fibre;

FIG. 7 shows one embodiment of the second class of embodiments where thenear confocal optical transmission means is provided by the cladding ofsingle mode optical fibre;

FIG. 8 shows a variation of the second class of embodiments usingcladding modes of a single mode fibre where the near confocal light isextracted from the side of the fibre;

FIG. 8A shows a variation of the embodiment of FIG. 8, but with a secondwaveguide providing an alternative path for the near confocal light tobe converged to the photodetector;

FIG. 8B shows a variation of the embodiment of FIG. 8A, but with thesecond fibre provided with a material of variable refractive index toprovide the variable selection means;

FIG. 9 shows another variation of the embodiment of FIG. 8;

FIG. 9A shows a variation of the second class with the near confocallight extracted progressively;

FIG. 10 shows detail of another embodiment of the second class using aball lens to angularly code the light before it enters the collectionend of the fibre;

FIG. 10A shows a variation of the embodiment of FIG. 10.

FIG. 11 shows an example of the third class embodiments using a gradientindex fibre and etched sections of fibre for the selection means;

FIG. 11A shows the gradient index profile of the fibre of FIG. 11;

FIG. 12 is a diagram showing an emission end of a gradient index fibreand selection means in embodiments of the third class which make use ofthe projected image of a fibre tip;

FIG. 13 shows a further embodiment in which a variable amount of thehigher order modes can be extracted by the selective positioning of apolymer block in contact with a polished portion of the optical fibre.

Referring now to FIG. 1, there is shown a schematic diagram of ray pathsfrom points in an object above and below the focal plane. Specifically,a single mode optical fibre in a fibre confocal microscope typically mayuse the core 10 of a single mode optical fibre to transmit laser sourcelight from a laser (not shown). At an end 11 of the core 10 of theoptical fibre, the laser light projects outward in a cone of divergenceangle approximately 8 to 10° for a typical fibre (exaggerated in thefigure) through a focusing lens 12, focusing the light to a pointobservational field P within an object to be observed (object notshown). Since the light returning through the focusing lens 12 and backinto the end 11 of the core of single mode fibre must pass through thepoint P, it is predominantly light emanating from point P whichre-enters the core, providing the desired isolation of the light fromthe focal plane F and enabling this "confocal" light to be collected bythe use of separations means such as a beamsplitter or optical fibrecoupler. Light from points Q and R in the vicinity of P but a distance Dabove and below respectively the focal plane F is not focused into theend of the core 11, but has a focal point either immediately in front ofor behind the end of the core 11. As a result, at the front face 14 ofthe optical fibre, light from points Q and R diffusively impinges on thecladding material of the fibre. Normally the cladding of a single modefibre is surrounded by a jacket 13 having a refractive index greaterthan that of the cladding and therefore inhibiting the propagation ofrays called cladding modes through the cladding, called cladding modes.

Clearly, light emanating from points closer than a distance D fallswithin a circle of radius R at the face 14 of the optical fibre, and asD is increased, so does R increase. Accordingly, the distance R from theaxis at which light impinges on the optical fibre is related to adistance D from the focal plane from within which the light hasemanated.

This well-known aperture relationship is what allows the opening andclosing of the pinhole in a standard confocal microscope to provideincreased depth of field.

Accordingly, if the light can be transmitted in such a way that thisdistance relationship is preserved or otherwise encoded, then the lightis transmitted in a separable manner such that when it reaches an exitregion of the fibre, it may by various means be selected in aprogressive manner to define the equivalent of a variable pinhole.

In the first class of preferred embodiments, which are the simplest tovisualise, this distance relationship is preserved by providing aplurality of isolated channels, as shown in FIG. 2. For example, asshown in FIG. 2a this may be realised by a coherent fibre bundle, withthe laser light delivery and confocal return fibre 20 at its centre, anda plurality of collection fibres surrounding the delivery core.Alternatively, a multi-core fibre may be used such as the "four leafclover" design shown in FIG. 2b, or a multiple clover design shown inFIG. 2c. An alternative variation involves concentric cylindrical waveguide regions as shown cut-away in FIG. 2d, separated by lowerrefractive index regions 23 (for example, silica glass) which space thehigher refractive index cylinders by a distance sufficient to reducecoupling between the cylinders to an acceptable level over the lengthsused in the fibre optic patch cords. A jacket 24 encompasses the fibre.It may be desirable for the outer cylindrical wave guide structures tobe thicker than inner ones.

Referring now to FIG. 3, there is shown a means of tapping the nearconfocal light in a progressive manner to provide the variable selectionmeans of the first class, applicable to the four leaved clover fibrearrangement. An etched region 30 of the fibre exposes a channel 31 andis filled with optical cement 32 having a refractive index equal to orgreater than that of silica. Within the optical cement 32 is embedded aphoto detector 33 as a part of the detector means. Light travellingalong channel 31 encounters the etched region 30 and is diverted intothe optical cement, activating the photo detector 33. Similarly, asecond etched region 34 is provided which exposes a section of a secondone 35 of the four leaved clover channels, and is again filled withoptical cement 36 containing a second photo detector 37. The variableselection means is provided by switching means, and hardware orsoftware, to select light from the desired detectors. Further down thefibre, a launch mode stripper 38 is provided which exposes all but thecentral core and prevents laser light in the light beam from the laserfrom travelling down the four leaved clover cores and into the detectors33 and 37.

At a remote end 39 of the fibre, light from the confocal core emergesand returns through light source focussing optics 390 and is partiallydeflected by a light separator in the form of beam splitter 391 into aphotomultiplier tube 392 to provide detection of the confocal returnlight, in a similar manner to known laser scanning confocal microscopes.

The embodiment shown in FIG. 3 uses only one single mode fibre core forboth transmission of the light beam from the laser to the object and forcollection and transmission to the detector of the confocal light(emanating from the confocal plane). Scanning can be achieved, as isdisclosed in U.S. Pat. No. 5,120,953, by a number of means, includingvibration of the fibre tip, and/or conventional scanning mirrors betweenthe fibre tip and the specimen. Similar embodiments can be envisaged, inaccordance with the disclosures of U.S. Pat. No. 5,120,953, whichinvolve a separate fibre for transmission of the light beam. All thedescriptions given here including the embodiment of FIG. 3 correspond toembodiments in which the light is conveyed to the microscope head andspecimen by means of a core of a single mode fibre. In these embodimentsthe alternative channels for conveying the near confocal light back tothe photodetector are within the same fibre which conveys the light tothe specimen. In accordance with the teachings of U.S. Pat. No.5,120,953, a beamsplitter may be used and the fibre conveying theconfocal light back to the photodetector may be a second completelyseparate fibre. All the methods described in the current specificationcan also be applied to the two fibre system in which the modal selectionmeans are applied to the second return fibre to selectively extend orrestrict the depth of field.

Referring now to FIG. 4, there is shown a similar variable selectionmeans for the concentric wave guide structure shown in FIG. 2d, withetched regions of progressively deeper extent being applied along anexit region of the fibre. The first region 40 extracts light only fromthe outermost core. The next region 41 is slightly deeper and itextracts light from the second outermost core, light from the outermostcore already having been extracted. Further and deeper regions may bearranged in succession. A launch mode stripper (not shown) is providedat the end of the fibre, as in FIG. 3, and similar switching means areprovided.

Referring now to FIG. 5, there is shown an alternative means ofproviding variable selection means in the first class of embodiments.The exit region is provided by an emission end 50, the same end whichreceives the light beam 51 from the laser 52. The laser focusing optics53 also acts for the return light as a near confocal focussing means toprovide an enlarged projected image of an emission end 50 of the fibreat a remote point 54. An iris diaphragm 55 is used to progressivelyexclude the selected portions of the light from entering thephotomultiplier tube (not shown). This method is applicable to any ofthe isolated channel arrangements shown in FIG. 2. For isolated channelarrangements other than the concentric waveguide structure of FIG. 2(d),the preservation of XY information could also be used to advantage if amultiple photo detector is used. For example, if a quadrant photodetector is used in association with the four leaf clover design, adifference between the outputs of the four channels can be used indisplaying other imaging modes such as differential interferencecontrast.

Referring now to FIG. 6 there is shown near confocal light exemplifiedby rays 61 and 62 propagating as cladding modes through the cladding ofa single mode fibre with core 63 accepting the confocal light 64. Singlemode fibres are composed of a Ge doped core 63, typically of about 3 μmdiameter, surrounded by silica cladding 65 of lower refractive indexthan the core, the diameter of the cladding typically being about 125μm. Surrounding the cladding is a jacket. In such an arrangement, thecladding modes are accepted and propagate by total internal reflectionif they are incident on the collection end 41 at an angle of less thanabout 30°. If it is desired to allow the cladding modes to propagate,the jacket should be constructed from a material of lower refractedindex than the cladding. Transparent silicone rubber is a suitablematerial. Normally, the jacket is constructed from acrylic materialwhich inhibits the propagation since cladding modes are normallyundesirable.

In a microscope of conventional dimensions and using 125 micron fibre,the cladding 65 cannot be used on its own to transmit the near confocallight in a separable manner such that a selected portion of the nearconfocal light emerging from one or more selected distances within arange of distances above and below the focal plane may be separated fromthe remainder of the near confocal light. This is firstly because thelight rays are mixed as they propagate through the cladding and emergeat the other end of the fibre in a disordered fashion, such that theordered relationship between distance from the optical axis of entry anddistance range from the focal plane is lost. Secondly, the angle ofincidence of the near confocal light will not vary sufficiently whencollected by 125 micron fibre in a manner dependent on depth of field.

However, the fact that the angle of exit of the rays is always equal tothe angle of entry can be used to advantage by the addition of opticalelement on or near the fibre tip. Referring now to FIG. 7, there isshown part of the optics of one of the second class of embodiments whichuses the cladding modes by coding the distance from the axis intoincidence angle of propagation within the cladding. Rays emerging fromthe confocal point P are shown entering the single mode core at 70(exaggerated angle). The collection end 71 of the fibre is fashionedinto a curved shape to provide a lensing effect which bends rays to agreater extent the more distant they enter from the optical axis. Theconfocal light effectively enters the core end 70 without refraction,since the curved shape is behind the core end 70. This shape may bemanufactured by heating the end of the fibre so that softening andsurface tension produces a curved shape. Light emerging from points Sand T progressively farther from the focal plane enters along rays S'and T' progressively farther from the optical axis, and thereforecorresponds to rays S" and T" of increasing angle of propagation. Jacket71a is composed of a suitable low RI material such as silicone plastic.At an emission end 72 of the fibre, the distance from the axis at whichthe rays S" and T" emerge is not ordered in accordance with the angle ofpropagation, but the angle of emergence is so ordered. This can be usedto advantage by provision of far-field iris diaphragm 73 in front of thenear confocal focusing means 74, which also may act as the laserfocussing optics. In a fully opened position the iris diaphragm admitssubstantially all of the light emerging from the emission end 72, whichis then focused onto an image of the fibre tip at detection focal plane75. A further near-field iris diaphragm 76 in front of the detectionfocal plane 75 will not operate in a progressive manner similar to irisdiaphragm 73 since the spatial variation of intensity in a projectedimage of the fibre end is not correlated in this embodiment withdistance from the axis of entering light, but may be used to exclude thenear confocal light from entering the photo detector when operating atmaximum resolution to detect only confocal light. When the far-fieldiris diaphragm 73 is partially opened, the near-field iris diaphragm 76will vary the proportion of near confocal light being admitted.

It may be advantageous to provide a section near the tip of the fibrehaving reduced overall diameter (not shown) by hydrofluoric acid etchingor other techniques so that the radius of curvature of the tip can bedecreased to give a reduced path length for the required separation ofthe near confocal light. This section may be reduced in diameter in asingle step or gradually as an adiabatic taper.

Referring now to FIG. 8, there is shown an alternative means ofextracting the near confocal light from one of the second class ofembodiments which uses cladding mode propagation. A glass block 80 isoptically connected by optical glue 81 to an exposed part of thecladding of the fibre. The refractive index of the glass block must behigher than or equal to the refractive index of the optical glue whichmust be higher or equal to the refractive index of the fibre cladding.Lens 83 focuses the light emerging through variable iris 84 ontophotomultiplier tube 85. A mirror 86 reflects light emerging from theother side of the fibre to follow substantially the same path. Acladding mode stripper 87 prevents laser light from the laser travellingalong the cladding. The confocal return light travelling along the coreof the fibre, which passes through the centre of lens 83 is extracted atthe fibre end in a standard manner and passed via beamsplitter 89 tophoto detector 88. In fluorescence imaging applications, where thewavelength of the object emanated light differs from the wavelength ofthe laser source light, a laser exclusion filter 890 can be used toexclude any stray laser source light which is reflected from the tipback into the fibre as cladding modes. Anti-reflection coatings or othertip treatments could be employed without filter 890 if the apparatus isto be used in reflection mode confocal microscopy.

An alternative similar arrangement is shown in FIG. 8A, where a secondglass fibre 8A1 , preferably of larger diameter and the same or higherrefractive index as the cladding of the first fibre 8A2 (correspondingto the fibre of FIG. 8), is fused to the first fibre 8A2 over a lengthof some millimeters. The light travelling down the cladding of firstfibre 8A2 is channelled into the larger second fibre 8A1 in proportionto the cross-sectional areas of the two fibres, and the angular orderingof the light rays is maintained. If a 500 micrometer fibre is used forthe second fibre 8A1, and a 125 micrometer fibre for first fibre 8A2,then approximately 94 percent of the cladding modes will be channelledinto the second fibre 8A1. Lens 8A3 and iris diaphragm 8A4 may then bepositioned remote from fibre 8A2, removing the need for encompassing thelens around fibre 8A2.

FIG. 8B shows another alternative means of providing the variableselection means, where a material 8B1 of variable refractive index iscontacted with an exposed part 8B2 of the cladding of the fibre(corresponding to the fibre of FIG. 8). A variable amount of higherorder modes can then be extracted through surface 8B2 and discarded. Theremaining complementary fraction passes to photomultiplier tube 8B3. Thematerial of variable refractive index may be a collection of differentliquids selectively being made to contact the surface 8B2, or a seriesof soft polymer blocks.

A further alternative similar arrangement is shown in FIG. 9 where aperspex box 90 surrounds the fibre, including a region containingexposed cladding at 92. Clear polyester resin 93 is poured into the box90 and sets.

Referring now to FIG. 9A, there is shown an alternative exit region forembodiments of the second class. Rather than have a single exit regionas in the embodiments of FIGS. 8 and 9, whereby the selection means isprovided by lenses and irises, it is possible to use successive regionsof the fibre with the jacket 9A1 removed and drops of optical glue withsuccessively increasing refractive index to cause rays of successivelylower angle of internal propagation to be extracted. If the cladding 9A2typically has a refractive index of 1.45 and the jacket 9A1 typicallyhas a refractive index of 1.40, in a first stripped region a blob 9A4 ofoptical glue may have a refractive index of, for example, 1.41 toextract a first portion of high angle propagation rays into the glue inwhich is placed a photo detector 9A3. At a second region optical glue9A5 having refractive index of 1.42 and detector 9A6 extracts furtherlight greater than a lower angle, and so on. As in the embodiments shownin FIGS. 3 and 4, launch mode strippers are disposed at one end andswitching means provide the variable selection means (not shown). Theblobs 9A4 and 9A5 are not to scale and are typically 3 to 4 mm or morein size, sufficiently long in an axial direction of the fibre to extendat least as far as the internally reflecting ray path "pitch".

A conventional objective lens may be used in place of the curved fibretip if the light is allowed to propagate on an extended path to allowsufficient lateral divergence of the near confocal light from theconfocal light cone to thereby produce the required coding of lateralseparation into angular separation. The advantage of the curved fibreend is that it allows for a much shorter distance between fibre tip andspecimen. Any arrangement where the confocal channel is disposed suchthat the confocal light is not adversely refracted by the lens may besuitable to allow short distance separation between tip and specimen.

The integral focusing provided by the curved end 71 in FIG. 7 may beprovided by separate small lens glued onto the fibre such as a ball lens100 shown in FIG. 10, typical ray paths 101 for which are shown. Adisadvantage of this embodiment is that the confocal light and the laseremission light is also focussed by the ball lens, again requiring alarger distance between fibre tip and specimen. This embodiment alsodoes not correct for chromatic aberration. However, one advantage isthat relative movement between fibre tip and lens is made possible. Inorder to match the projected laser beam to a variety of lenses in themicroscope turret, each having a different back focal diameter it isdesirable to have an adjustment mechanism by means of which the fibretip entering the head can be moved towards or away from the lensadjacent the fibre tip. FIG. 10A shows lens 10A1 attached by a flexibleoptical glue 10A3 to fibre tip 10A2 housed within a piezoelectriccylinder 10A4. The cylinder 10A4 is contractible in length, which shiftsthe fibre tip longitudinally by a few microns. This motion increases thewidth of the light beam with negligible alteration in the beam angle,and after passing through the transfer optics this adjustment can beused to match the aperture of the objective lens being used, maximisingthe optical efficiency and resolution for each lens.

One of a third class of embodiments is shown in FIG. 11 where instead ofa lens on or in front of the face of the fibre, a gradient index"mammary profile" fibre is used in place of a single mode fibre. In thisfibre, the optical material surrounding the single mode core hasgradations of refractive index, providing a curved ray paths 110 for themodes propagating in the fibre outside the core. FIG. 11A shows arefractive index profile for the "mammary profile" fibre. Regions 11A1correspond to the polymer jacket, regions 11A2 to the glass cladding,region 11A3 to the multimode-supporting region of gradations of indexand 11A4 to the single mode core region. The maximum angle ofpropagation of ray paths is related to the distance from the opticalaxis at which the ray path enters as a minimum distance of approach ofthe ray to the outer face 111. Thus, rays entering at a greater distancefrom the optical axis have greater maximum angle of propagation as theycross the optical axis and go closer to the outer face 111.

Thus progressive selection of the near confocal light by means of aseries of progressively deeper etched regions with optical glue at 112a,112b and 112c is possible. The diagram in FIG. 11 of ray paths isschematic only. The rays in fact do not enter the fibre in a mannerwhich would cause the maintenance of node regions more than a shortdistance along the fibre. Each etched region is in fact not placedstrategically with respect to a node, but is elongate along the fibreaxis for about 3 to 4 mm, being several times the pitch length of theoscillatory ray path, whereby a single etched region on one side of thefibre to a depth Δ will absorb effectively all light rays which comewithin Δ of the surface at their maximum of oscillation.

Referring now to FIG. 12, there is shown the emission end 120 of agradient index fibre according to another variation of the third class.A double iris diaphragm arrangement similar to that shown in FIG. 7 isemployed here, although the principle of operation is somewhatdifferent. The light emerging from the remote end of the fibre is eitherfar from the axis and has a low angle of incidence such as rays 121 and122, or is near to the axis and has a high angle of incidence such asrays 123 and 124. The light which proceeds to iris diaphragm 126 islight which is emerging from the fibre at a low angle, aperture 125blocking out high angle light. This light is approximately ordered indistance from the core in a corresponding manner to the light enteringthe fibre, in turn corresponding to distance from the focal plane in thespecimen. For example, ray 121 is shown emerging slightly further fromthe core than ray 122, and is excluded from detection by the variableiris diaphragm 126, while ray 122 is accepted. The situation is morecomplex than this in practice, because the idealisation of nodesdepicted in FIG. 11 is not realised. This results in there being alsorays ordered in angle rather than distance form the core, and these maybe progressively selected by operation of iris diaphragm 125. Inpractice, there is a continuous range of intermediate cases also. Theentire range can however be progressively selected to an acceptabledegree by operating iris diaphragm 125 and 126 simultaneously. Theprojected image of the fibre tip at iris diaphragm 126 is thendisplacement-coded from the axis in the desired manner, and itsoperation of the iris diaphragm 126 is such as to produce a variablepinhole effect for the near field modal pattern rays. Operated inconjunction with variable occlusion of the for field modal pattern raysby iris diaphragm 125 this will provide operation which is functionallyequivalent to a conventional variable physical pinhole in a confocalmicroscope system.

Still another arrangement in shown in FIG. 13 where a first fibre 131 isshown cast into a polymer block 132, the surface 133 having beenpolished away to expose the cladding almost to the core 134. A variableamount of the higher order modes can then be extracted through surface133 and discarded by sliding a second polymer block 135 progressivelyover the surface 133, as is known analogously in variable ratio fibrecoupler technology.

In order to achieve appropriate separation of confocal and near confocallight, it may be necessary in many embodiments of the invention to usebeam extenders in order to provide an adequate distance between thefibre tip and the objective while maintaining manageable productdimensions. This can be designed in a compact package using standardopposing mirror techniques as is well known in the art.

The principle of coupling out modes from a fibre by means of asurrounding medium, the refractive index of which can be changed, mightalso be applied to the light form the laser on the way to the microscopehead. If a few moded communications fibre was used as the opticaltransmission means and the cladding glass was etched away from a sectionof this fibre and replaced with a controllable variable RI material thenthe modes passing into the microscope could be controlled at the sametime as the modes coming back to the detectors. This would have certainadvantages in giving extra signal strength for low fluorophoreconcentrations where there is fluorescence saturation and where nonlinear bleaching may be a problem.

Modifications may be made to the invention as would be apparent to aperson skilled in the art of confocal optical design. For instance, theinvention is not restricted to applications requiring adiffraction-limited confocal spot and imaging systems other thanmicroscopes which can make use of the same optical principles are withinits scope. Further still, the near-field iris diaphragms which aredisposed adjacent a projected image of the fibre end and its associatedphoto detector may be replaced by CCD arrays if desired and theselective exclusion of light performed in software. CCD arrays maysimilarly be used with far-field pattern decoding.

Also, a number of embodiments have been shown which variously use exitregions in either a mid region of the near confocal return fibre or anend; selection means which may be classified as "near field" or "farfield", being composed of lenses and irises or switching means; and"coding" systems in three classes using isolated channels or angularcoding. Other combinations of these basic ideas may be envisaged and arealso within the scope of the invention.

Further, as explained above the single-fibre embodiment shown here canbe replaced by dual fibre systems, with source fibre and return fibresbeing separate or also with the confocal return being provided by aseparate fibre to the near-confocal return. These and othermodifications may be made without departing from the ambit of theinvention, the nature of which is to be ascertained from the foregoingdescription and the drawings.

We claim:
 1. A confocal imaging system comprising:a light source forsupply of a light beam; light focusing means for focusing light from thebeam onto a point observational field on or within an object and forreceiving object emanated light emanating from the vicinity of the pointobservational field; a detector for detecting the object emanated light;scanning means operable to cause relative movement between the objectand the point observational field such that the point observationalfield scans over a focal plane transverse to an optical axis of theimaging system; and flexible optical transmission means for transmittingthe source light beam from the light source to the light focusing meansand for transmitting the object emanated light to the detector, andhaving light separator means to separate the object emanated light fromthe light beam for passing to the detector and confocal opticaltransmission means to transmit the object emanated light emerging onlyfrom the point observational field;wherein the optical transmissionmeans further comprises(i) flexible near confocal optical transmissionmeans having a light collection end adjacent to a light collection endof the confocal optical transmission means and adapted to transmit onlynear confocal light emerging from points in the object located within arange of distances above and below the focal plane in such a manner thata selected portion of the near confocal light emerging from greater thana corresponding selected distance within said range is substantiallyseparable from the remainder; (ii) an exit region for exit of at least aportion of said near confocal light from the flexible near confocaloptical transmission means; and wherein there is further providedvariable selection means to define said selected portion and exclude itfrom the detector.
 2. A confocal imaging system as claimed in claim 1wherein the near confocal optical transmission means comprises a widediameter fibre or the cladding of a single mode optical fibre.
 3. Aconfocal imaging system as claimed in claim 2 wherein the focussingmeans causes rays entering the light collection end of the optical fibreto be transmitted through the fibre at an angle which increases with thedistance of a point of entry of the ray into the collection end from theoptical axis of the fibre such that the substantial separability of saidselected portions is thereby attained.
 4. A confocal imaging system asclaimed in claim 3 wherein the focussing means comprises a ball lensglued onto the collection end of the optical fibre.
 5. A confocalimaging system as claimed in claim 3 wherein the focussing meanscomprises the collection end of the fibre fashioned into a curved shapeto provide a lensing effect which bends rays to a greater extent themore distant they enter from the optical axis.
 6. A confocal imagingsystem as claimed in claim 5 wherein there is provided a narrow sectionnear the tip of the fibre having reduced overall diameter such that theradius of curvature of the tip is decreased to give a reduced pathlength for the required separation of the near confocal light.
 7. Aconfocal imaging system as claimed in claim 6 wherein the narrow sectionis reduced in diameter in a single step.
 8. A confocal imaging system asclaimed in claim 6 wherein the narrow section is reduced in diameteradiabatically.
 9. A confocal imaging system as claimed in claim 3wherein the variable selection means includes a variable diaphragmdisposed adjacent the exit region to exclude light emerging at greaterthan a selected angle.
 10. A confocal imaging system as claimed in claim2 wherein the exit region is provided by an emission end of the fibre.11. A confocal imaging system as claimed in claim 10 wherein thevariable selection means includes near confocal focussing means to focusan image of the emission end of the fibre onto a second variablediaphragm.
 12. A confocal imaging system as claimed in claim 3 whereinthe exit region is provided by one or more regions where the side of thefibre is exposed and contacts an extracting material with refractiveindex suitably matched to the fibre so as to extract some or all of thenear confocal light.
 13. A confocal imaging system as claimed in claim12 wherein the exit region is provided by a single such exposed region.14. A confocal imaging system as claimed in claim 13 wherein theextracting material is a glass block optically connected to the exposedregion.
 15. A confocal imaging system as claimed in claim 13 wherein aclear box surrounds the fibre, including the exposed region and theextracting material is a clear resin set inside the box to opticallyconnect with the exposed region.
 16. A confocal imaging system asclaimed in claim 13 wherein the variable selection means includes avariable diaphragm disposed adjacent the exit region of the opticalfibre to exclude light emerging at greater than a selected angle.
 17. Aconfocal imaging system as claimed in claim 12 wherein the exit regionis provided by a plurality of said exposed regions arranged along thefibre contacting materials having progressively greater refractive indexto progressively extract rays of lower angle, the variable selectionmeans comprising optical or electronic switching means.
 18. A confocalimaging system as claimed in claim 2 wherein the near confocal opticaltransmission means comprises a gradient index fibre.
 19. A confocalimaging system as claimed in claim 18 wherein the exit region isprovided by successively deepening etched areas in the fibre side withcorresponding photo detectors.
 20. A confocal imaging system as claimedin claim 18 wherein the exit region is provided by an emission end ofthe fibre.
 21. A confocal imaging system as claimed in claim 20 whereina first variable diaphragm is provided to admit only low angle lightthrough near confocal focussing means to project an image of the fibretip onto a second variable diaphragm in front of the detector.
 22. Aconfocal imaging system as claimed in claim 1 wherein the near confocaloptical transmission means comprises a plurality of optically isolatedchannels having adjacent ends at said light collection end to providesaid substantially separable transmission.
 23. A confocal imaging systemas claimed in claim 22 wherein the plurality of channels is provided bya bundle of optical fibres.
 24. A confocal imaging system as claimed inclaim 22 wherein the plurality of channels is provided by a largediameter optical fibres with a plurality of cores.
 25. A confocalimaging system as claimed in claim 24 wherein the plurality of channelsis a plurality of coaxial concentric waveguides, mutually separated byoptically insulating material.
 26. A confocal imaging system as claimedin claim 22 wherein the exit region of the near confocal opticaltransmission means is provided by a plurality of etched sections offibre exposing different ones or subsets of said plurality of channelsand containing optical cement to divert light travelling in thecorresponding one or subset of channels to corresponding photodetectors.27. A confocal imaging system as claimed in claim 26 wherein thevariable selection means comprises switching means to select output fromdifferent ones or subsets of said photodetectors.
 28. A confocal imagingsystem as claimed in claim 22 wherein the exit region is provided byopposite ends of the plurality of isolated channels forming an emissionend of the fibre or fibre bundle.
 29. A confocal imaging system asclaimed in claim 28 wherein the variable selection means comprisesfocusing means to project an image of the emission end onto a regioncontaining a variable diaphragm to progressively exclude from detectionsaid selected portion, the detector being disposed behind the diaphragm.30. A confocal imaging system as claimed in claim 1 wherein the confocaloptical transmission means is integral with the near confocaltransmission means.
 31. A confocal imaging system as claimed in claim 30wherein the confocal optical transmission means comprises a single modecore disposed inside the near confocal optical transmission means.
 32. Aconfocal imaging system as claimed in claim 1 wherein the confocaloptical transmission means is separate from the near confocal opticaltransmission means.